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1.
Anal Chem ; 95(4): 2203-2212, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36669833

RESUMO

Antibody combination therapies have become viable therapeutic treatment options for certain severe diseases such as cancer. The co-formulation production approach is intrinsically associated with more complex drug product variant profiles and creates more challenges for analytical control of drug product quality. In addition to various individual quality attributes, those arising from the interactions between the antibodies also potentially emerge through co-formulation. In this study, we describe the development of a widely applicable multi-dimensional liquid chromatography coupled to tandem mass spectrometry method for antibody homo- versus hetero-aggregate characterization. The co-formulation of trastuzumab and pertuzumab was used, a challenging model system, comprising two monoclonal antibodies with very similar physicochemical properties. The data presented demonstrate the high stability of the co-formulation, where only minor aggregate formation is observed upon product storage and accelerated temperature or light-stress conditions. The results also show that the homo- and hetero-aggregates, formed in low and comparable proportions, are only marginally impacted by the formulation and product storage conditions. No preferential formation of hetero-aggregates, in comparison to the already existing pertuzumab and trastuzumab homo-aggregates, was observed.


Assuntos
Anticorpos Monoclonais , Espectrometria de Massas em Tandem , Cromatografia Líquida , Anticorpos Monoclonais/química , Trastuzumab/química
2.
Carbohydr Polym ; 270: 118338, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34364593

RESUMO

Ulvans from green algae are promising compounds for plant protection because they are environmentally friendly and induce plant defense responses. We analyzed the structure-function relationship of ulvan polymers and oligomers for their elicitor activity in suspension-cultured cells of three dicot species. The polysaccharide from Ulva fasciata was characterized regarding its monosaccharide composition, degree of sulfation, and molecular mass. The polymer was partially depolymerized using acid hydrolysis, and the oligomers were separated using size exclusion chromatography. The oligomeric fractions were analyzed revealing mostly sulfated and de-sulfated ulvan dimers. Both the polymer and the oligomer fractions induced an NADPH oxidase-dependent oxidative burst in plant cells. The elicitor activity of the ulvan dimers did not require sulfation. By identifying the smallest elicitor-active unit, HexA-Rha, we took an important next step to understand how the structure influences ulvan elicitor responses. The desulfated ulvan dimer is discussed as a promising agro-biologic for sustainable agriculture.


Assuntos
Polissacarídeos/química , Ulva/química , Clorófitas/química , Cromatografia em Gel/métodos , Hidrólise , Peso Molecular , Oligossacarídeos/química , Oxirredução , Imunidade Vegetal , Polímeros/química , Explosão Respiratória , Ulva/metabolismo
3.
Bioinformatics ; 36(22-23): 5330-5336, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33325487

RESUMO

MOTIVATION: Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. RESULTS: Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae. AVAILABILITY AND IMPLEMENTATION: The source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating systems. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

4.
Antibodies (Basel) ; 8(4)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31581521

RESUMO

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure-function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

5.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180048

RESUMO

A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seeds by a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circular dichroism spectrum showed that the protein contains approx. 39% beta-sheets but only approx. 5% alpha-helices. The protein is thermally quite stable, and exhibits a cooperative thermal unfolding transition at approx. 70 degree C, as determined by circular dichroism spectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS) allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identity with a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 and Cys156 was also detected. The protein showed approx 24 and approx 25% sequence identity with alpha-amylase/subtilisin inhibitor from barley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structural fold similar to other Kunitz-type TIs. The presence of Cys149-Cys156 disulfide bond as detected by MS and a second disulfide bond connecting Cys44-Cys91, conserved in all Kunitz-type TIs, is also identified in the model.


Assuntos
Abelmoschus/química , Peptídeos/química , Proteínas de Plantas/química , Sementes/química , Tripsina/química , Abelmoschus/metabolismo , Sequência de Aminoácidos , Sulfato de Amônio/química , Sítios de Ligação , Cromatografia/métodos , Eletroforese em Gel de Gradiente Desnaturante , Modelos Moleculares , Peso Molecular , Peptídeos/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Sementes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Termodinâmica
6.
Int J Biol Macromol ; 134: 921-935, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31078591

RESUMO

Hot-water soluble polysaccharides H-1-3 and H-2-1 were isolated from the thalli of the lichen Xanthoria parietina (L.) Th. Fr. and purified by ion exchange and gel permeation chromatography. Structure elucidation was mainly based on 2D-NMR and nano-ESI-Q-TOF MS/MS experiments. H-1-3 (13.7 kDa) was shown to be linear α-glucan with α-d-Glcp-(1 → [→[4)-α-d-Glcp-(1]2 → [6)-α-d-Glcp-(1]3 → 4)]n core backbone. The (1,4)- and (1,6)-α-d-Glcp linkages were in a 2:3 M ratio. H-2-1 (525 kDa) was characterized as a complex branched ß-galacto-α-mannan with →[6)-α-d-Manp-(1 → [2,6)-α-d-Manp-(1]2 → [2)-α-d-Manp-(1]2→]n core units and main side chains of (1,3)-ß-d-Galf linked at O-6 to →2)-α-d-Manp-(1→, together with minor terminal units of 1,4/1,6-α-D -Glcp units attached to the core chain at O-6 position and α-L-Rhap linked to Galf side chain at O-2 position (Manp: Galf: Glcp: Rhap linkage ratio = 9:3:2:1). H-2-1 exerted strong immunoactivity in vitro and activated murine RAW macrophages 264.7 towards significantly increased phagocytosis, TNF-α and IL-1ß secretion. These effects are due to an interaction of the galactomannan with the transmembrane pattern-recognition protein Dectin-2 of the macrophages.


Assuntos
Líquens/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Animais , Galactose/análogos & derivados , Glucanos/química , Hidrólise , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Espectroscopia de Ressonância Magnética , Mananas/química , Camundongos , Peso Molecular , Fagocitose , Polissacarídeos/isolamento & purificação , Células RAW 264.7 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
Int J Biol Macromol ; 127: 649-656, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30708015

RESUMO

Conversion of crystalline chitin to soluble sugar molecules, using lytic polysaccharide mono-oxygenases (LPMOs) has emerged as a new avenue for the production of biofuels. The present study describes the role of accessory domains in a multi-modular LPMO from Bacillus thuringiensis serovar kurstaki (BtLPMO10A). The full length BtLPMO10A (BtLPMO10A-FL) possesses an N-terminal LPMO of AA10 family (BtAA10) and a C-terminal CBM5 (BtCBM5) connected via two fibronectin (Fn) III domains (aligned as AA10-FnIII-FnIII-CBM5 from N- to C-terminus). To determine the role of individual domains, we generated truncation mutants of BtLPMO10A-FL. Substrate binding and kinetic studies revealed that BtCBM5 was involved in increasing binding efficiency of BtAA10 which otherwise has feeble binding towards ß-chitin and could not bind to α-chitin. Furthermore, binding assays also indicated that the presence of CBM5 increases the binding efficiency of BtLPMO10A-FL under extreme pH conditions. FnIII domains neither bind nor assist BtLPMO10A-FL in chitin binding and serve as linkers in BtLPMO10A-FL. BtLPMO10A-FL and BtAA10 generated oxidized chito-oligosaccharides from the insoluble ß-chitin substrate. It is concluded that BtCBM5 is responsible for increasing binding efficiency of BtLPMO10A-FL, whereas; BtAA10 domain is accountable for oxidative cleavage of recalcitrant chitin.


Assuntos
Bacillus thuringiensis/enzimologia , Proteínas de Bactérias/química , Quitina/química , Oxigenases de Função Mista/química , Oligossacarídeos/química , Domínio Catalítico , Cristalografia por Raios X , Oxirredução , Especificidade por Substrato
8.
Fitoterapia ; 129: 226-236, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30031897

RESUMO

Lichenan (molecular weight 275 kDa, ß-D-1,3/1,4-glucopyranose ratio 1:3) from Cetraria islandica at a concentration of 100 µg/mL induced terminal cellular differentiation of primary human keratinocytes as demonstrated by immunofluorescence staining using cytokeratin 10 and involucrin as marker proteins. Lichenan-derived oligosaccharides (DP3 to 8), obtained by acid-catalyzed partial hydrolysis of the polymer, did not influence cellular differentiation. Cytokeratin, filaggrin, involucrin, loricrin and transglutaminase gene expression as typical differentiation markers was increased by lichenan in a time-dependent manner. Lichenan upregulated gene cluster which were mostly related to cellular differentiation with focus on MAPK signaling as was shown by Whole Human Genome Microarray. These gene expression data from the array experiments were subsequently confirmed by qPCR for selected genes. For identification of the molecular binding structures of lichenan 1- and 2-D PAGE of keratinocyte protein membrane preparations was performed, followed by blotting with FITC-labeled lichenan and subsequent mass spectrometric identification of the pinpointed proteins. Epidermal growth factor receptor (EGFR) and integrin ß4, both proteins being strongly involved in induction of keratinocyte differentiation were identified. In addition, protein disulfide isomerase A3 (PDIA3) showed strong binding to FITC-lichenan, indicating this enzyme to be an intracellular target of the glucan for induction of the cellular differentiation of keratinocytes. As lichenan did not influence the EGFR phosphorylation and the phosphorylation of CREB transcription factor but strongly interacted with cytosolic proteins it is hypothized that the glucan may interact with EGFR and is subsequently internalized into the cell via endosomal uptake, interacting with PDIA3, which again alters TGFß1 signaling towards keratinocyte differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Glucanos/farmacologia , Queratinócitos/efeitos dos fármacos , Parmeliaceae/química , Células Cultivadas , Proteínas Filagrinas , Humanos , Queratinócitos/citologia
9.
Anal Chem ; 90(15): 8989-8997, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-29939014

RESUMO

Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) as a human pathogenic subgroup of STEC are characterized by releasing Stx AB5-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes, Stx1a and Stx2a are of eminent clinical importance in human infections being associated with life-threatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with a generalized fatal outcome for the animals. Binding toward the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here, we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS), (iii) functional Stx-receptor real-time interaction analysis employing the surface acoustic wave (SAW) technology, and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a, and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of life-endangering Stx subtypes at the protein level.


Assuntos
Edematose Suína/microbiologia , Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/microbiologia , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/citologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Chlorocebus aethiops , Humanos , Separação Imunomagnética/métodos , Viabilidade Microbiana , Escherichia coli Shiga Toxigênica/química , Som , Suínos , Células Vero
10.
Int J Biol Macromol ; 110: 588-597, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29341923

RESUMO

PP2-like chitin binding phloem exudate lectins, abundant in the sieve tube of cucurbits, have been implicated to play key roles in wound sealing and antipathogenic responses of the plant. Here we report the affinity purification, macromolecular characterization and carbohydrate binding properties of a new chitooligosaccharide-specific lectin from the phloem exudate of ivy gourds (Coccinia indica). The protein, CIA24, has a subunit mass of 24 kDa. Partial sequence analysis indicated that CIA24 exhibits high homology with CIA17 and other Cucurbitaceae PP2 proteins whereas CD spectroscopic studies suggested that ß-sheets constitute the predominant secondary structure. Temperature dependent CD spectroscopic and differential scanning calorimetric studies revealed that CIA24 is a highly thermostable protein, which undergoes complete unfolding at ∼105 °C. Isothermal titration calorimetric studies suggested that binding of chitooligosaccharides to CIA24 is a highly exothermic process. The lectin combining site can accommodate upto a tetrasaccharide with the binding stoichiometry (n) close to unity with respect to each protein subunit, whereas for chitohexaose a sharp decrease in the binding stoichiometry (n) to ∼1:0.5 was observed. This suggests that the protein probably undergoes dimerisation in presence of chitohexaose, wherein two protein molecules bind to the oligosaccharides from the reducing and non-reducing end, respectively.


Assuntos
Quitina/análogos & derivados , Cucurbitaceae/química , Floema/química , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Quitina/química , Quitina/metabolismo , Quitosana , Cucurbitaceae/metabolismo , Oligossacarídeos , Floema/metabolismo , Lectinas de Plantas/metabolismo , Ligação Proteica , Especificidade por Substrato
11.
Int J Biol Macromol ; 108: 1227-1236, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29126937

RESUMO

Phloem protein-2 (PP2) is an abundant soluble protein in the sieve elements in plants. Its lectin property was reported in various species. The primary structure of a 17kDa PP2 from Coccinia indica (Coccinia indica agglutinin, CIA17), determined by mass spectrometry, shows extensive homology with PP2 super family phloem lectins. Analysis of mass spectrometric data indicated the presence of 16 potential allelic variants of CIA17 with insignificant divergence in the primary structure. The primary structure contains an intramolecular disulfide bridge between Cys-34 and Cys-51, which is conserved across various cucurbit species and hence likely to be important for carbohydrate binding. CD spectroscopic studies revealed that CIA17 is rich in antiparallel ß-sheets, similar to PP2 proteins from Cucurbita maxima and Arabidopsis thaliana. CD spectra recorded at various temperatures showed very little change in the spectral intensity and shape up to 90°C, suggesting that CIA17 is a highly thermostable protein. Atomic force microscopic studies revealed that CIA17 forms filamentous structures at higher concentrations. In light of these results, we propose that CIA17 and other PP2 proteins play a role in the plant defense against pathogens by directly binding with the chitin cell wall, and also promote wound healing by forming self-assembled filaments.


Assuntos
Aglutininas/química , Aglutininas/isolamento & purificação , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Agregados Proteicos , Sequência de Aminoácidos , Cromatografia de Afinidade , Peso Molecular , Análise de Sequência
12.
Anal Chem ; 89(5): 2893-2900, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28192919

RESUMO

Partially acetylated chito-oligosaccharides (paCOS) have diverse bioactivities that turn them into promising compounds especially for medical and agricultural applications. These properties likely arise from different acetylation patterns, but determining the sequences of paCOS and producing paCOS with patterns of interest have proven difficult. We present a novel method for sequencing submicrogram amounts of paCOS using quantitative mass spectrometry, allowing one to rapidly analyze the substrate specificities of chitosan hydrolases that can be used to produce paCOS. The method involves four major steps: (i) acetylation of free amino groups in paCOS using a deuterated reagent; (ii) labeling the reducing end with an 18O-tag; (iii) quantifying paCOS using [13C2, 2H3]-labeled isotopologs as internal standards; (iv) sequencing paCOS by tandem MS. Eventually, this method will aid in developing enzymes with cleavage patterns optimized for producing paCOS with defined patterns of acetylation and specific bioactivities.

13.
Arch Biochem Biophys ; 609: 59-68, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27664852

RESUMO

An α-D-galactose specific lectin belonging to the family of jacalin-related lectins (JRL) has been purified by affinity chromatography on cross-linked guar-gum. Mass spectrometric data revealed that the protein harbors two chains like all the members of galactose-specific jacalin-related lectins (gJRL). De novo sequencing of proteolytic peptides demonstrated that the heavier chain consists of 133 amino acids and the lighter chain comprises of 21 or 24 amino acids. The heavier chain contains one N-glycosylation site (Asn47) occupied with either pauci-mannose type [GlcNAc2(Fuc)Man3(Xyl)] or complex type [GlcNAc2(Fuc)Man3(Xyl)GlcNAc(Fuc)Gal] N-glycans. Circular dichroism spectroscopy indicated that the secondary structure of the lectin is predominantly made up of ß-sheets, and differential scanning calorimetry revealed a thermal denaturation temperature of 77.6 °C. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assays on MCF-7 and MDCK cells showed that the lectin is highly cytotoxic towards both cell lines when dosed at micromolar concentrations, suggesting that it may play a role in the defense mechanism of the plant.


Assuntos
Galactose/química , Morus/química , Lectinas de Plantas/química , Animais , Varredura Diferencial de Calorimetria , Cromatografia de Afinidade , Dicroísmo Circular , Cães , Feminino , Humanos , Células MCF-7 , Células Madin Darby de Rim Canino , Espectrometria de Massas , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína
14.
Cell Microbiol ; 18(10): 1339-48, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26990252

RESUMO

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34(+) haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a-mediated cellular injury of developing erythrocytes. CD34(+) haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a-binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx-mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx-producing pathogens.


Assuntos
Escherichia coli Êntero-Hemorrágica/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Toxina Shiga/farmacologia , Sobrevivência Celular , Células Cultivadas , Eritrócitos/microbiologia , Eritrócitos/fisiologia , Hematopoese/imunologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/microbiologia , Humanos
15.
Methods Mol Biol ; 1394: 163-179, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26700048

RESUMO

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. As a result of their higher hydrophilicity N-glycopeptides comprising short peptide backbones are preferably accumulated by the ZIC-HILIC-based separation procedure. By employing this approach complications associated with low ionization efficiencies of N-glycopeptides resulting from signal suppression in the presence of highly abundant nonglycosylated peptides can be largely reduced. Here, we describe a simple protocol aimed at the enrichment of N-glycopeptides derived from in-solution and in-gel digestions of SDS-PAGE-separated glycoproteins preceding mass spectrometric analysis.


Assuntos
Cromatografia/métodos , Glicopeptídeos/química , Glicoproteínas/química , Espectrometria de Massas/métodos , Glicosilação , Interações Hidrofóbicas e Hidrofílicas , Proteólise
16.
Biomacromolecules ; 16(7): 2157-67, 2015 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-26068019

RESUMO

Xyloglucan XG (molecular weight 462 kDa, 1,4-/1,4,6-(pGlc) linked backbone, side chains of 1-pXyl, 1,2-pXyl, 1-p-Gal) was isolated from the seeds of Tropaeolum majus. XG (100 µg/mL) induced terminal cellular differentiation of human keratinocytes, as demonstrated by immunofluorescence staining and Western blot using cytokeratin 10 and involucrin as marker proteins. Differentiation was also induced by XG-derived oligosaccharides (degree of polymerization 7-9). Quantitative real-time polymerase chain reaction (qPCR) revealed the induction of gene expression of typical differentiation markers (cytokeratin, filaggrin, involucrin, loricrin, transglutaminase) in a time-dependent manner. Whole human genome microarray indicated that most of upregulated genes were related to differentiation processes. Microarray findings on selected genes were subsequently confirmed by qPCR. For identification of the molecular target of xyloglucan PAGE of keratinocyte membrane preparations was performed, followed by blotting with fluorescein isothiocyanate-labeled XG. XG interacting proteins were characterized by MS. Peptide fragments of epidermal growth factor receptor (EGFR) and integrin ß4 were identified. Subsequent phospho-kinase array indicated that phosphorylation of EGFR and transcription factor cAMP response element-binding protein (CREB) was decreased in the XG-treated cells. Thus, the XG-induced differentiation of keratinocytes is proposed to be mediated by the inhibition of the phosphorylation of EGFR, leading to a dimished CREB activation, which is essential for the activation of cellular differentiation.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Glucanos/farmacologia , Queratinócitos/efeitos dos fármacos , Tropaeolum/química , Xilanos/farmacologia , Diferenciação Celular , Células Cultivadas , Proteínas Filagrinas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Fosforilação/efeitos dos fármacos , Sementes/química
17.
Nanomedicine ; 11(2): 275-82, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25461290

RESUMO

The cytokine secretion of primary cells of human macrophages during the interaction of TiO2 nanoparticles (with an average primary size of 100-120 nm) is investigated down to concentration levels suggested to be relevant for in vivo conditions. We find that high TiO2 concentrations induce the cytokines Interleukin IL-1ß, IL-6, and IL-10 secretion, while at low concentrations only IL-6 secretion is observed. To obtain further evidence on in vivo conditions we investigated the development and structure of the protein corona of the nanoparticles. We demonstrated that the surface of TiO2 particles attract preferably secondary modified proteins which then induce cytokine secretion of macrophages. Our results indicate that concentration of corona covered TiO2 particles below 1 µg/ml induce IL-6 secretion which is reported to be responsible for the development of autoimmune diseases as well as for the secretion of acute phase proteins. FROM THE CLINICAL EDITOR: This study investigates the effects of protein corona covered titanium dioxide nanoparticles on human macrophages, concluding that concentration of such particles below 1 µg/ml induces IL-6 secretion, which may be responsible for the development of autoimmune diseases as well as for the secretion of acute phase proteins. This finding has important implications on future applications of such nanoparticles.


Assuntos
Interleucina-8/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Nanopartículas/administração & dosagem , Titânio/administração & dosagem , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/biossíntese , Nanopartículas/química , Tamanho da Partícula , Titânio/química , Fator de Necrose Tumoral alfa/biossíntese
18.
Infect Immun ; 82(11): 4631-42, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25156739

RESUMO

Enterohemorrhagic Escherichia coli (EHEC), a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno- and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associated E. coli (HUSEC) clones, but they lacked virulence markers typical of uropathogenic E. coli. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenic E. coli (ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressed stx genes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urine in vitro by typical uropathogenic E. coli isolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated.


Assuntos
Cistite/microbiologia , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/microbiologia , Adulto , Idoso , Animais , Linhagem Celular , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Feminino , Humanos , Camundongos , Adulto Jovem
19.
J Biol Chem ; 289(40): 27585-603, 2014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-25157101

RESUMO

α-Neurexins (α-Nrxn) are mostly presynaptic cell surface molecules essential for neurotransmission that are linked to neuro-developmental disorders as autism or schizophrenia. Several interaction partners of α-Nrxn are identified that depend on alternative splicing, including neuroligins (Nlgn) and dystroglycan (αDAG). The trans-synaptic complex with Nlgn1 was extensively characterized and shown to partially mediate α-Nrxn function. However, the interactions of α-Nrxn with αDAG, neurexophilins (Nxph1) and Nlgn2, ligands that occur specifically at inhibitory synapses, are incompletely understood. Using site-directed mutagenesis, we demonstrate the exact binding epitopes of αDAG and Nxph1 on Nrxn1α and show that their binding is mutually exclusive. Identification of an unusual cysteine bridge pattern and complex type glycans in Nxph1 ensure binding to the second laminin/neurexin/sex hormone binding (LNS2) domain of Nrxn1α, but this association does not interfere with Nlgn binding at LNS6. αDAG, in contrast, interacts with both LNS2 and LNS6 domains without inserts in splice sites SS#2 or SS#4 mostly via LARGE (like-acetylglucosaminyltransferase)-dependent glycans attached to the mucin region. Unexpectedly, binding of αDAG at LNS2 prevents interaction of Nlgn at LNS6 with or without splice insert in SS#4, presumably by sterically hindering each other in the u-form conformation of α-Nrxn. Thus, expression of αDAG and Nxph1 together with alternative splicing in Nrxn1α may prevent or facilitate formation of distinct trans-synaptic Nrxn·Nlgn complexes, revealing an unanticipated way to contribute to the identity of synaptic subpopulations.


Assuntos
Encéfalo/metabolismo , Distroglicanas/metabolismo , Glicoproteínas/metabolismo , Neuropeptídeos/metabolismo , Processamento Alternativo , Animais , Distroglicanas/química , Distroglicanas/genética , Glicoproteínas/genética , Humanos , Ligantes , Camundongos , Neuropeptídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Sinapses/genética , Sinapses/metabolismo
20.
Int J Biol Macromol ; 69: 400-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24907509

RESUMO

An affinity purified mannose/glucose specific lectin from the seeds of Dolichos lablab (Indian bean/lablab bean) resolves into five subunits upon SDS-PAGE in the range of Mr 12-20kDa. Partial de novo sequencing of subunits resulted in 88% and 73% sequence coverage for α and ß subunits of the cDNA derived FRIL (Flt3 receptor interacting lectin) sequence, respectively and suggested that four bands correspond to the α-subunits while the band of lowest molecular mass is designated as ß. It was proposed in an earlier study on FRIL that the difference in molecular mass of α-subunits is due to differences in C-terminal processing and differential N-glycosylation i.e. numbers of N-glycans present (Colucci et al., 1999). Thus, differential N-glycosylation of the purified mannose/glucose specific lectin was unravelled by in-gel trypsin/chymotrypsin digestion of the α-subunits followed by desalting and ZIC-HILIC enrichment of N-glycopeptides. Subsequently, analyses by nano electrospray ionisation quadrupole time of flight mass spectrometry and low-energy collision-induced dissociation experiments revealed the presence of a typical paucimannose type N-glycan (Man2(Xyl)GlcNAc2(Fuc)) in α subunits 2-4.


Assuntos
Dolichos/química , Glucose/química , Manose/química , Lectinas de Plantas/química , Polissacarídeos/química , Sementes/química , Sequência de Aminoácidos , Glicopeptídeos/química , Glicosilação , Dados de Sequência Molecular
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